A CTL-based vaccine or vaccine component is considered important for eliciting immunity that contains disease, and perhaps for preventing infection with HIV-1. Thus far, only one CTL-based vaccine has reached advanced human trials, but the results were disappointing. Although it is unclear why the vaccine failed, evidence suggests that immunogenicity was adequate to elicit some protective CTL responses. We hypothesize that the shortcoming was not the delivery system, but rather the sequences being delivered. Antigenic exposure to whole proteins mimics infection with whole virus, which is a naturally failing process. We further hypothesize that misguided early immunodominance against variable epitopes is the central mechanism for natural failure of CTL in HIV-1 infection. To address this issue, we propose vaccine sequences that come from highly conserved and highly expressed proteins, to focus initial CTL responses against epitopes with these properties. We also propose that the vaccine be polyvalent to represent the remaining sequence variability in these proteome regions, which is similar intra-clade and inter-clade. Thus the vaccine represents the variability of HIV-1 both across and within clades, dealing with variability to which a person could be exposed, as well as variability that can develop within a person (escape mutation). The vaccine sequences will be delivered using DNA-prime and recombinant Ad5 boost, given that Ad5 appeared immunogenic in the STEP trial and this strategy remains a viable vaccine candidate in humans. We aim 1) to create immune-focusing vaccine constructs that deliver conserved and highly expressed regions of the HIV-1 proteome and represent inter-and intra-clade variability of HIV-1;2) to confirm the immunogenicity of our vectors in a standard DNA-prime and rAd5-boost vaccination strategy in rhesus macaques;3) to test the immune-focusing abilities of our vaccine constructs in rhesus macaques using crossover prime-boost vaccination;and 4) to test the immune-focusing abilities of our Env vaccine construct in rhesus macaques using SHIV 89.6P infection. PUBLIC HEALTH RELEVANCE: The need for an effective HIV-1 vaccine needs little introduction. While treatment regimens have advanced enormously in efficacy and reduced side effects, lifelong treatment remains an impractical global solution due to expense, long term toxicities, poor healthcare infrastructure in developing countries, and increasing drug resistance. Historically, effective vaccines have been the best approach to dealing with global epidemics.